The ability to select the sex of offspring produced in fertilization has long been recognized as having tremendous commercial potential and application. An excellent example of this may be found in the field of animal husbandry.
Dairy farm operations require the female of the species, cows, for milk production. Males of the species, bulls, are only important for breeding purposes. Thus, it is clear that any method that safely and effectively increases the production of female offspring versus male offspring would greatly benefit dairy cattle breeders and farmers and, therefore, be very valuable. Such a method would allow more effective and efficient farm operation, save money and increase farm milk production.
Of course, it should be recognized that humans may also benefit from such a method of selecting the sex of offspring. Parents could very simply satisfy their desires to have a son or daughter. This allows improved family planning, may reduce family tension and also limit family size for the benefit of all involved.
Numerous attempts have been made in the past to separate x-(male determining) and y-(female determining) bearing spermatozoa in order to determine the sex of the offspring before conception. The methods used include centrifugation, filtration, electrophoresis, immunological techniques and exposure of spermatozoa to alkaline or acidic environments.
Mechanical methods of separating male and female determining spermatozoa, based on the difference in density between the two types, are disadvantageously characterized by reduced spermatozoa survivability and by reduced viability of the surviving spermatozoa. Immunological methods of spermatozoa separation do not suffer as dramatically from these disadvantages.
U.S. pat. no. 4,448,767 to Bryant discloses two methods of immunological spermatozoa separation. The first method is a single antibody separation system wherein male specific antibody is bound to sephadex beads placed in a column. Native or unseparated spermatozoa is then added to the column. The male determining spermatozoa become bound to the male specific antibody/Sephadex bead conjugates as the female spermatozoa are eluted from the column. The male determining spermatozoa are then recovered separately.
The second method is a dual antibody separation system wherein native sperm is treated with male specific antibody. A second antibody, capable of specifically binding the male specific antibody is coupled to Sephadex beads placed in a column. The male specific antibody treated spermatozoa is then added to the column. The male determining spermatozoa become bound to the second antibody/Sephadex bead conjugates through the male specific antibody as the female spermatozoa are eluted from the column. The male determining spermatozoa are then recovered separately as in the first method.
While the two Bryant methods do serve to increase the percentage of mammalian offspring of either sex as desired, the methods are not without their disadvantages and, therefore, may be improved. Specifically, Bryant discloses a complicated technique of animal immunization with histocompatability (H-Y) antigen and multiple repeated absorptions through female spleen cells to separate and purify the male specific antibody used in the Bryant method. Despite this difficult and tedious technique, it should be appreciated that the resulting antibody is still not completely binding specific to the H-Y antigen found only in male determining and not female determining spermatozoa. Thus, it should be appreciated that a portion of the resulting male specific antibody used in Bryant could disadvantageously bind through antigens other than the y-antigen to some female determining spermatozoa. As a result, some female determining spermatozoa are bound to the Sephadex beads in the Bryant columns and eluted with the male determining spermatozoa fraction. Thus, it is clear that the highest levels of effective separation are not attainable with the Bryant method.
Bryant also disadvantageously requires the packing of a column with antibody/Sephadex bead conjugate. The column must be packed properly so that the rate of flow or spermatozoa elution is within a range allowing complete immunoreaction between the spermatozoa and the antibody of the antibody/bead conjugates. Too fast a flow rate means incomplete immunoreaction and separation and, therefore, the presence of male determining spermatozoa in the eluted female determining spermatozoa fraction. The proper packing of the column requires a great deal of preparation time and large quantities of expensive eluant or filtration solution are required to complete the separation and recover the majority of the spermatozoa from the column. This last consideration is particularly important when you consider that different eluants are typically required for each species of spermatozoa being separated. Thus, large quantities of a number of different and sometimes expensive eluants must be stored at the separation lab.
An additional problem in Bryant relates to the utilization of protein containing media for spermatozoa dilution prior to exposure of the spermatozoa to the antibody. Disadvantageously, the protein in the media retards the cross reactivity of the spermatozoa and the antibody. It is believed that the existing protein fraction in the spermatozoa diluent coats the spermatozoa covering the histocompatability H-Y antigen sites on the male determining spermatozoa and preventing antibody/antigen binding. This causes incomplete separation as, with either method in Bryant, coated male determining spermatozoa are eluted with the female determining spermatozoa.
From the above, it should be appreciated that a need exists for an improved method of immunologic spermatozoa separation.